WO2018233596A1 - 体外敲除T细胞中靶基因的方法以及该方法中使用的crRNA - Google Patents

体外敲除T细胞中靶基因的方法以及该方法中使用的crRNA Download PDF

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WO2018233596A1
WO2018233596A1 PCT/CN2018/091804 CN2018091804W WO2018233596A1 WO 2018233596 A1 WO2018233596 A1 WO 2018233596A1 CN 2018091804 W CN2018091804 W CN 2018091804W WO 2018233596 A1 WO2018233596 A1 WO 2018233596A1
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gene
crrna
cell
cells
targeting
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PCT/CN2018/091804
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English (en)
French (fr)
Chinese (zh)
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彭作翰
沈连军
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN201880004422.2A priority Critical patent/CN109963944A/zh
Priority to MX2019014516A priority patent/MX2019014516A/es
Priority to RU2020100919A priority patent/RU2020100919A/ru
Priority to AU2018288048A priority patent/AU2018288048A1/en
Priority to KR1020207000299A priority patent/KR20200018572A/ko
Priority to EP18820610.6A priority patent/EP3650545A4/en
Priority to CA3064807A priority patent/CA3064807A1/en
Priority to US16/623,605 priority patent/US20200181608A1/en
Priority to JP2019570534A priority patent/JP2020528738A/ja
Publication of WO2018233596A1 publication Critical patent/WO2018233596A1/zh

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Definitions

  • the invention belongs to the field of biomedicine. Specifically, it relates to a method for knocking out a target gene in a T cell in vitro, a crRNA used in the method, and a T cell obtained by the method and use thereof.
  • Adoptive cell therapy which involves the transfer of autologous antigen-specific T cells produced ex vivo, is a promising strategy for the treatment of viral infections and cancer.
  • T cells used in adoptive immunotherapy can be generated by amplification of antigen-specific T cells or by genetically designed T cell redirection (Park, Rosenberg et al. Trends Biotechnol. 2011, 29(11): 550-557).
  • CART is an isolated T cell that is genetically engineered into a specific antigen receptor (CAR) to enhance the targeting, killing activity and persistence of T cells, and the recognition of tumor cell surface antigens is not dependent on MHC.
  • CARs consist of extracellular antigen binding regions, transmembrane regions, and signal transduction regions of intracellular T cell receptors (such as CD3 ⁇ and costimulatory molecules).
  • the extracellular antigen binding region is composed of a light chain variable region (VL) and a heavy chain variable region (VH) of a monoclonal antibody, and is hinged to form a single chain fragment variable (scFv), which is capable of recognizing a specific Tumor antigen.
  • CAR has a better therapeutic effect in patients with lymphoma who are ineffective in other treatments.
  • the CART-19 study conducted by Carl June of the University of Pennsylvania showed that in 75 patients with leukemia (including adults and children), 45 patients had complete remission after CART cell therapy.
  • Cellectis has successfully eradicated multiple cases of relapsed acute lymphoblastic leukemia (ALL) by directional knockout of TCR ⁇ gene (reduced GVHD) and CD52 gene (resistance of cells to alemtuzumab) by TALEN technology. child.
  • ALL relapsed acute lymphoblastic leukemia
  • CD52 resistance of cells to alemtuzumab
  • Cellectis knocking out TCR through TALEN requires a cumbersome build process and large-scale sequencing.
  • CRISPR-associated, CRISPR-Cas9 achieves editing of genes by recognizing specific DNA sequences and is simpler and more efficient than TALEN.
  • the object of the present invention is to overcome the problems existing in the prior art in immunotherapy, and to provide a method for constructing TCR-negative T cells, which knocks out TCR by CRISPR/Cas9 gene editing technology, and provides TCR negative T obtained by the method. cell.
  • Another object of the present invention is to provide a TCR, B2M and PD1 triple negative T cell and a method of constructing the same.
  • TCR-negative T cells TCR and PD-1 or B2M double-negative T cells and TCR/B2M/PD1 triple-negative T cells are sorted by magnetic beads, and are used for adoptive cell immunotherapy of tumors and the like.
  • a method of knocking out one or more target genes in a T cell in vitro comprising the steps of:
  • RNP protein RNA complex
  • sgRNA directs the Cas9 protein to a target sequence of the corresponding target gene, respectively, and the target Sequence hybridization wherein the target gene is cleaved, and wherein the cleavage efficiency of the target gene is greater than 75%.
  • the target gene is selected from one or more of the TRAC, TRBC, B2M and PD1 genes,
  • the sgRNA targets a coding sequence of the target gene or a regulatory sequence thereof.
  • the sgRNA is sequentially sequenced from 5' to 3' by a 17-20 nt target target gene of crRNA and Cas9 protein Corresponding tracrRNAs are ligated, wherein the length of the crRNA is preferably 17 nt.
  • the oligodeoxyribonucleic acid in the method of knocking out one or more target genes in T cells in vitro, is double-stranded DNA having a length of 100-250 bp or a length of 100 -250 nt of single-stranded DNA.
  • the crRNA targeting the TRAC gene is selected from the group consisting of the crRNAs shown in SEQ ID NOs: 1-12 Any one or more of the same, the crRNA sequence targeting the B2M gene is set forth in SEQ ID NO: 13, and the crRNA targeting the PD1 gene is selected from any one of the crRNAs shown in SEQ ID NOs: 14-16. Kind or more.
  • the Cas9 protein is a Cas9 protein from Streptococcus pyogenes, the sequence is SEQ ID NO: 18. Shown.
  • the Cas9 protein in the method of knocking out one or more target genes in T cells in vitro, is a Cas9 protein from Streptococcus pyogenes, and the tracrRNA corresponding to the Cas9 protein The sequence is shown in SEQ ID NO: 17.
  • the T cells are selected from the group consisting of helper T cells, cytotoxic T cells, memory T cells, Regulatory T cells, natural killer T cells, ⁇ T cells, CAR-T cells, and TCR-T cells.
  • the present invention provides a target gene knockout T cell obtained according to the above method.
  • the invention provides a crRNA for knocking out a targeted gene, wherein the crRNA comprises one or more sequences selected from the group consisting of SEQ ID NOs: 1-16.
  • the present invention is for knocking out a coding gene of a target gene, a coding sequence of a targeted gene, or a regulatory sequence thereof, wherein the targeted gene is selected from the group consisting of TRAC, TRBC, B2M, and One or more of the PD1 genes.
  • the invention is for knocking out a crRNA of a targeted gene, wherein the targeting gene is a TRAC gene, and the crRNA is selected from one of SEQ ID NOs: 1-12 or Multiple.
  • the invention is for knocking out a crRNA of a targeted gene, wherein the targeting gene is a B2M gene, and the crRNA sequence is set forth in SEQ ID NO: 13.
  • the invention is for knocking out a crRNA of a targeted gene, wherein the targeting gene is a PD1 gene, and the crRNA is selected from one of SEQ ID NOs: 14-16 or Multiple.
  • the present invention also provides an sgRNA for knocking out a targeting gene, the sgRNA being formed by linking a crRNA to a tracrRNA corresponding to a Cas9 protein, wherein the crRNA comprises one or more selected from the group consisting of SEQ ID NO: 1.
  • the sequence of -16 is a sequence of -16.
  • the invention is for knocking out a sgRNA of a targeted gene, wherein the targeting gene is selected from one or more of the TRAC, TRBC, B2M and PD1 genes.
  • the present invention is for knocking out a sgRNA targeting a gene TRAC, which is formed by linking a crRNA to a tracrRNA corresponding to a Cas9 protein selected from the group consisting of SEQ ID NO: 1-12 One or more of the ones shown.
  • the invention is used to knock out a sgRNA targeting the gene B2M, which is formed by the tracrRNA junction of the crRNA and the Cas9 protein, as shown in SEQ ID NO: 13.
  • the present invention is for knocking out an sgRNA targeting the gene PD-1, which is formed by linking a crRNA to a tracrRNA corresponding to a Cas9 protein selected from the group consisting of SEQ ID NO: 14. One or more of the -16.
  • the invention is for knocking out a sgRNA of a targeted gene, wherein the Cas9 protein is a Cas9 protein from S. pyogenes as set forth in SEQ ID NO: 18.
  • the invention is for knocking out a sgRNA targeting a gene, wherein the tracrRNA sequence corresponding to the Cas9 protein is set forth in SEQ ID NO: 17.
  • the invention provides a kit for gene knockout comprising:
  • a one or more crRNAs as described above, or one or more sgRNAs as described above;
  • c oligodeoxyribonucleic acid or fish sperm DNA fragment.
  • the oligodeoxyribonucleic acid in the kit for gene knockout, is double-stranded DNA of 100-250 bp in length or single-stranded DNA of 100-250 nt in length. .
  • the Cas9 protein is a Cas9 protein from Streptococcus pyogenes, and the tracrRNA sequence corresponding to the Cas9 protein is SEQ ID NO: 17. Shown.
  • the invention provides the use of a knockout T cell of the invention for the preparation of an anti-tumor drug.
  • the present invention also provides the use of the knockout T cells of the present invention for the preparation of a medicament for controlling infectious diseases caused by viruses or bacteria.
  • TCR, B2M or PD1 are effectively knocked out using the designed crRNA and method.
  • the in vitro killing activity of CART cells after TCR and B2M and/or PD1 knockout is not affected by TCR, B2M and/or PD1 gene knockout.
  • FIG. 1 Comparison of knockout efficiency for different delivery systems. The results show that the RNP delivery mode has the highest gene knockout efficiency in Jurkat cells.
  • FIG. 2A-2B Effect of N-oligo on the knockdown efficiency of T cell genes based on the CRISPR-Cas9 system.
  • Figure 2A is a comparison of gene knockout efficiencies in T cells;
  • Figure 2B is a comparison of gene knockout efficiencies in CART cells.
  • Figure 3 Effect of fish sperm DNA fragments on T cell gene knockout efficiency.
  • Figure 5 Detection of the knockdown effect of the PD1 gene by the screened crRNA.
  • Figures 6A-6B Analysis of gene mutations caused by RNP and N-Oligo or fish sperm DNA.
  • Fig. 6A is an analysis result for TRAC
  • Fig. 6B is an analysis result for B2M.
  • Figures 7A-7C RNP off-target rate analysis.
  • Fig. 7A shows the results of the off-target analysis of the TRAC gene;
  • Fig. 7B shows the results of the off-target analysis of the B2M gene;
  • Fig. 7C shows the results of the off-target analysis of the PD1 gene.
  • Figures 8A-8B Analysis of CD25 and CD69 activation of TRAC knockout T cells.
  • Figure 8A is a comparison of CD69 activation and
  • Figure 8B is a comparison of CD25 activation.
  • the invention provides a method of altering a target gene in a cell.
  • the studies described herein demonstrate the use of the allele targeting approach of the CRISPR/Cas system to produce mutant cells with efficiencies as high as 80%.
  • the work described herein surprisingly and unexpectedly demonstrates a multiplex guide strategy that provides a method for specifically identifying useful RNA leader sequences, as well as for targeting specific genes (eg, TRAC, TRBC, B2M) , PD1) specific boot sequence.
  • An exemplary method of altering a polynucleotide sequence of a target gene in a cell comprises correlating the polynucleotide sequence with a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequence (Cas)
  • the protein is contacted with one or two ribonucleic acids to form an RNP, wherein the ribonucleic acid directs the Cas protein to a target motif of a polynucleotide sequence of the target gene and hybridizes to the target motif, wherein the target gene
  • the polynucleotide sequence was cleaved, and the efficiency of the cells in which the RNP was transformed was 75% or more.
  • the present invention contemplates altering the polynucleotide sequence of a target gene in any manner that is readily available to the skilled artisan using the CRISPR/Cas system of the present invention.
  • Any CRISPR/Cas system capable of altering the polynucleotide sequence of a target gene in a cell can be used.
  • a variety of Cas proteins can be used in this type of CRISPR/Cas system (Haft et al. PLoS Comput Biol. 2005; 1(6) e60).
  • Such Cas proteins allow the CRISPR/Cas system to alter the molecular sequences of the polynucleotide sequences of the target genes in the cell, including RNA binding proteins, endonucleases and exonucleases, helicases, and polymerases.
  • the CRISPR/Cas system is a CRISPR Type I system.
  • the CRISPR/Cas system is a CRISPR Type II system.
  • the CRISPR/Cas system of the invention can be used to alter the polynucleotide sequence of a target gene in a cell.
  • the present invention contemplates altering the polynucleotide sequence of a target gene in a cell for any purpose.
  • the polynucleotide sequence of a target gene in a cell is altered to produce a mutant cell.
  • the Cas9 protein herein may illustratively be a S. pyogenes Cas9 protein or a functional portion thereof.
  • the Cas9 protein is a Cas9 protein from any bacterial species or a functional portion thereof.
  • the Cas9 protein is a member of the Type II CRISPR system, which typically includes a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 protein.
  • CRISPR-derived RNA CRISPR-derived RNA
  • tracrRNA trans-activating RNA
  • the alterations modifies the polynucleotide sequence of the target gene from an undesired sequence to a desired sequence.
  • the CRISPR/Cas system of the invention can be used to correct for any type of mutation or error in the polynucleotide sequence of a target gene.
  • the CRISPR/Cas system of the invention can be used to insert a nucleotide sequence that is missing in the polynucleotide sequence of a target gene due to deletion.
  • the CRISPR/Cas system of the present invention can also be used to delete or excise a nucleotide sequence due to an insertion mutation from a polynucleotide sequence of a target gene.
  • the CRISPR/Cas system of the invention can be used to replace an incorrect nucleotide sequence with a correct nucleotide sequence (eg, to restore a polynucleotide sequence of a target gene that is compromised by loss of a functional mutation)
  • the function ie SNP
  • the CRISPR/Cas system of the present invention can unexpectedly cleave target genes with high efficiency compared to conventional CRISPR/Cas systems.
  • the cleavage efficiency of the target gene is at least about 5%. In certain embodiments, the cleavage efficiency of the target gene is at least about 10%. In certain embodiments, the cleavage efficiency of the target gene is from about 10% to about 80%. In certain embodiments, the cleavage efficiency of the target gene is from about 30% to about 80%. In certain embodiments, the cleavage efficiency of the target gene is from about 50% to about 80%. In some embodiments, the target gene has a cleavage efficiency of greater than or equal to about 75%, or greater than or equal to about 80%.
  • the target gene is a genome. In some embodiments, the target gene is a human genome. In some embodiments, the target gene is a mammalian genome. In some embodiments, the target gene is a vertebrate genome.
  • a polynucleotide sequence or a portion thereof that knocks out a target gene using the CRISPR/Cas system of the present invention can be applied to various applications.
  • a polynucleotide sequence that knocks out a target gene in a cell can be performed in vitro for research purposes.
  • a polynucleotide sequence that knocks out a target gene in a cell can be useful for treating or preventing a disorder associated with expression of a polynucleotide sequence of the target gene (eg, by ex vivo knockout of cells)
  • the allele is mutated and those cells comprising the knockout mutant allele are introduced into the subject).
  • the invention provides a method of treating or preventing a condition associated with expression of a polynucleotide sequence in a subject.
  • the term "contacting" ie, contacting a polynucleotide sequence with a clustered regularly spaced short palindromic repeat (Cas) protein and/or ribonucleic acid
  • contacting is intended to include incubating the Cas protein in vitro and/or RNA or contact cells in vitro.
  • the step of contacting the polynucleotide sequence of the target gene with the Cas protein and/or ribonucleic acid as disclosed herein can be carried out in any suitable manner.
  • the cells can be treated in the form of adherent or suspension culture.
  • cells contacted with Cas protein and/or ribonucleic acid as disclosed herein may also be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environment, to stabilize or render the cells. Further differentiation.
  • the term "treating" or the like includes subjecting the cell to any type of process or condition, or performing any type of operation or procedure on the cell.
  • the term is directed to an individual providing a cell in which the polynucleotide sequence of the target gene has been altered ex vivo according to the methods described herein.
  • the individual is typically ill or injured, or is at an increased risk of illness relative to the average member of the population and requires such attention, care or management.
  • treating refers to administering to a subject an effective amount of a polynucleotide having a polynucleotide sequence that is altered ex vivo according to the methods described herein, such that the subject has the disease.
  • a reduction in at least one symptom or an improvement in the disease for example, a beneficial or desired clinical outcome.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the disease, stabilization of the disease state (ie, no deterioration), delay or reduction in disease progression. Slow, improved or alleviated disease states, and relief (whether partial or total), whether detectable or undetectable.
  • Treatment may mean prolonging survival as compared to expected survival in the absence of treatment.
  • treatment includes prophylaxis.
  • treatment is "effective” in the event that the progression of the disease is reduced or stopped.
  • Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.
  • Those in need of treatment include those that have been diagnosed with a disorder associated with expression of a polynucleotide sequence, as well as those that may develop such a disorder due to genetic susceptibility or other factors.
  • mutant cell refers to a cell having a resulting genotype that is different from its original genotype.
  • mutant cells exhibit a mutant phenotype, such as when a functionally normal gene is altered using the CRISPR/Cas system of the invention.
  • mutant cells exhibit a wild-type phenotype, such as when the CRISPR/Cas system of the invention is used to modify a mutant genotype.
  • the polynucleotide sequence of a target gene in a cell is altered to modify or repair the gene mutation (eg, to restore the normal genotype of the cell).
  • the polynucleotide sequence of a target gene in a cell is altered to induce a genetic mutation (eg, to disrupt the function of a gene or genomic element).
  • the alteration is an insertion deletion.
  • Insert deletion refers to a mutation resulting from an insertion, deletion or a combination thereof. As will be understood by those of skill in the art, unless the length of the insertion deletion is a multiple of three, an insertional deletion in the coding region of the genomic sequence will result in a frameshift mutation.
  • the alteration is a point mutation.
  • Point mutation refers to a substitution of one of the alternative nucleotides.
  • the CRISPR/Cas system of the invention can be used to induce insertion or point mutations of any length in a polynucleotide sequence of a target gene.
  • oligodeoxyribonucleic acid or “N-oligo” refers to a deoxyribonucleic acid fragment of a random sequence that is transformed into a cell together with RNP when a gene knockout is performed using an RNP delivery system, preferably a double length of 100-250 bp. Stranded DNA or 100-250 nt single-stranded DNA.
  • “Fish sperm DNA fragment” refers to a small molecule fragment in which a solution containing salmon sperm DNA is mechanically sheared to cut fish sperm DNA. For example, 1% salmon sperm DNA solution is repeatedly beaten with a 7-gauge needle to cut DNA into small molecules, and stored after dispensing.
  • knockout includes deletion of all or a portion of a polynucleotide of the target gene in a manner that interferes with the function of the polynucleotide of the target gene.
  • knockout can be achieved by altering the polynucleotide sequence of the target gene by inducing a functional domain of the polynucleotide sequence of the target gene in the polynucleotide sequence of the target gene (eg, Insertion deletion in the DNA binding domain).
  • cleavage of the target gene results in decreased expression of the target gene.
  • reduced is generally used herein to mean reducing a statistically significant amount.
  • reducing means reducing at least 10% compared to the reference level, for example by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, compared to the reference level, Or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 90%, or up to and including 100% reduction (ie, a level that is not present compared to the reference sample) , or any reduction between 10% and 100%.
  • statically significant refers to statistical significance and generally means two standard deviations (2SD) below or below the normal marker concentration.
  • 2SD standard deviations
  • the term refers to statistical evidence of the difference. It is defined as the probability of making a decision to reject a hypothesis when the hypothesis is actually true. The decision is often expressed using a p value.
  • cleavage of the target gene is cleavage of a homozygous target gene. In some embodiments, cleavage of the target gene is cleavage of a hybrid target gene.
  • the Cas9 protein (also known as CRISPR-related endonuclease Cas9/Csn1) is a polypeptide comprising 1368 amino acids.
  • An exemplary amino acid sequence of the Cas9 protein is set forth in SEQ ID NO: 18.
  • Cas9 contains two endonuclease domains, including the RuvC-like domain (residues 7-22, 759-766, and 982-989), which cleave target DNA that is not complementary to crRNA; and the HNH nuclease domain (residue) Base 810-872), which cleaves target DNA complementary to the crRNA.
  • T cell receptor is a heterodimeric protein receptor that presents a specific antigenic peptide on the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • TCR is a glycoprotein on the surface of a cell membrane in the form of a heterodimer formed by an alpha chain/beta chain or a gamma chain/delta chain.
  • the TCR heterodimer consists of alpha and beta chains in 95% of T cells, while 5% of T cells have a TCR consisting of gamma and delta chains.
  • the native ⁇ heterodimeric TCR has an alpha chain and a beta chain, and the alpha chain and the beta chain constitute a subunit of the ⁇ heterodimeric TCR.
  • each of the alpha and beta chains comprises a variable region, a junction region, and a constant region
  • the beta chain typically also contains a short polymorphic region between the variable region and the junction region, but the polymorphic region is often considered as a junction region. a part of.
  • Each variable region comprises three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimeric in framework regions.
  • the CDR regions determine the binding of the TCR to the pMHC complex, wherein the CDR3 is recombined from the variable region and the junction region and is referred to as the hypervariable region.
  • the alpha and beta chains of TCR are generally considered to have two "domains", namely a variable domain and a constant domain, and the variable domain consists of linked variable and linking regions.
  • the sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the constant domain sequence of the TCR molecule ⁇ chain is “TRAC*01”
  • the constant domain sequence of the TCR molecule ⁇ chain is “TRBC1*”. 01" or "TRBC2*01”.
  • the alpha and beta chains of TCR also contain a transmembrane and cytoplasmic regions with a short cytoplasmic region.
  • B2M also known as beta-2 microglobulin
  • B2M is the light chain of MHC class I molecules and is therefore an integral part of the major histocompatibility complex.
  • B2M is encoded by the b2m gene located on chromosome 15 as opposed to other MHC genes located on chromosome 6 as a cluster of genes.
  • the human protein consists of 119 amino acids and has a molecular weight of 11,800 Daltons.
  • a murine model of ⁇ -2 microglobulin deficiency has demonstrated that B2M is required for cell surface expression of MHC class I and stability of peptide binding channels.
  • PD-1 or "PD1” is a 50-55 kDa type I transmembrane receptor originally identified in a T cell line that undergoes activation-induced apoptosis. PD-1 is expressed on top of T cells, B cells and macrophages.
  • the ligand for PD-1 is the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC).
  • PD-1 is a member of the immunoglobulin (Ig) superfamily and contains a single IgV-like domain in its extracellular region.
  • the PD-1 cytoplasmic domain contains two tyrosines, of which the membrane closest to tyrosine (VAYEEL in mouse PD-1) is located within ITIM (the inhibitory motif of the immunoreceptor tyrosine).
  • ITIM the inhibitory motif of the immunoreceptor tyrosine
  • the presence of ITIM on PD-1 predicts that this molecule acts by recruiting cytosolic phosphatase to attenuate the signaling of antigen receptors.
  • the human and murine PD-1 proteins share approximately 60% amino acid identity with four potential N-glycosylation sites conserved and residues defining the Ig-V domain.
  • the ITIM-like motif around the ITIM and carboxy terminal tyrosine (TEYATI in humans and mice) in the cytoplasmic region is also conserved between human and murine orthologues.
  • PBMC separation tube Sepmate-50 (STEMCELL Technology), add 15 ml of Ficoll buffer (GE healthcare), and add a mixture of blood PBS.
  • PBS buffer containing 2% fetal bovine serum
  • the pellet was resuspended in PBS after centrifugation. For resuspended cell count, 10 ⁇ l of the suspension was added to 10 ⁇ l of 0.1% trypan blue to mix and count the cell number and survival rate.
  • the cell suspension was added to a 5 ml flow tube and placed in a magnetic pole for 5 minutes.
  • the cell suspension was quickly decanted, PBS buffer was added to the flow tube and resuspended, and repeated 3 times.
  • the obtained cell suspension was centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in MONZA VIVO-15 medium, and the density was adjusted to 1 ⁇ 10 6 /ml, and rIL-2 (R&D) was added thereto.
  • the concentration is 100 IU/ml. Then, it was cultured in a 37-degree cell culture incubator.
  • Anti-CD3/anti-CD28 magnetic beads (Life Technology) were resuspended in PBS buffer (containing 2 mM EDTA and 1% fetal bovine serum), and then placed in a magnetic pole for 2 minutes, and the supernatant was discarded. Repeat the above process 4 times. After the magnetic beads were taken, the number of magnetic beads was added to the purified T cells in a ratio of 1:1, mixed, and cultured at 37 degrees for 3 days. After 3 days, the magnetic beads were taken out, and the target cells were first resuspended several times with a pipette. The cell suspension was placed in a magnetic pole, and after standing for two minutes, the magnetic beads on the tube wall were discarded.
  • PBS buffer containing 2 mM EDTA and 1% fetal bovine serum
  • the CD19 CAR structure was constructed from CD19 scFv, Hinge structure, transmembrane structure, 4-1BB and CD3z, CD19 CAR and vector pHR-CAR.
  • the lentiviral plasmid pHR-CAR was ligated with the two helper plasmids dR8.91 and pCMV-VSV-G plasmids using the Tiangen Big Plasmid Kit.
  • Transfection system 1 Transfection system 2 pHR-CAR: 7.5 ⁇ g dR8.91: 5.625 ⁇ g pCMV-VSV-G: 1.875 ⁇ g Opti-MEM (Gibco): 700 ⁇ l Opti-MEM (Gibco): 700 ⁇ l P3000: 30 ⁇ l Lipofectamine: 36 ⁇ l
  • the human primary T cells were resuspended, placed in a magnetic pole for two minutes, and the cell suspension was taken. Cell suspension was performed on the cell suspension. After centrifugation of about 1 ⁇ 10 7 cells at 300 g for 5 minutes, the medium was discarded, and 1 ml of the new medium was added and resuspended. Add concentrated lentivirus to adjust the MOI to 5 and mix. After centrifugation at 2000 g for 90 minutes at 32 ° C, the supernatant was discarded, and the new medium (100 IU/ml of rIL-2) was added to adjust the cell density to 1 ⁇ 10 6 /ml. After resuspension, the newly isolated anti-- CD3/anti-CD28 magnetic beads. The cultivation was continued in a 37 ° C incubator. Obtain CAR-T cells.
  • a suitable target region was selected, and a 17-20 nt crRNA was designed.
  • the crRNA was ligated with the corresponding tracrRNA sequence of the Cas9 protein to form an sgRNA.
  • the crRNA with high knockout efficiency and low target rate was screened by experiment.
  • the selected partial crRNA sequences are as follows:
  • the Cas9 protein is from Cas9 Nuclease NLS (S. pyogenes (BioLabs)), corresponding to the tracrRNA sequence (SEQ ID NO: 17):
  • the amino acid sequence of the Cas9 (including NLS) protein used (SEQ ID NO: 18):
  • the sgRNA linked to the tracrRNA corresponding to the Cas9 protein described above was prepared as the crRNA shown in Table 1, and the crRNA was located at the 5' end of the tracrRNA.
  • DNA is obtained that can be used to transcribe sgRNA in vitro.
  • the obtained sgRNA was purified and detected by spectrophotometer and denaturing agarose gel electrophoresis, and all of them were ready to be dispensed immediately.
  • CAR-T cells which can also be used to knock primary T cells
  • the distribution conversion system 10 ⁇ l of Nucleofector buffer, 30 ⁇ g of Cas9 protein (about 9 ⁇ g/ ⁇ l) and 4 ⁇ g of sgRNA were mixed and incubated at room temperature for 10 minutes. After three days of activation, CAR-T cells were magnetically depleted of anti-CD3/anti-CD28 magnetic beads. 5 x 10 6 cells/tube were taken and centrifuged at 300 g for 5 minutes to completely remove the supernatant. Add the incubated electroporation system to the cell pellet, add 72 ⁇ l of Nucleofector buffer and 18 ⁇ l of Supply buffer, and mix and add to 100 ⁇ l of LONZA electroconversion cup.
  • CAR-T cells were cultured to day 10 after CRISPR-Cas9 knockout of TRAC, and TCR-negative cells were enriched. First centrifuge all cells: 300g for 5 minutes. Wash twice with PBS buffer (containing 2 mM EDTA and 1% fetal bovine serum). The cell density was adjusted to be 1 ⁇ 10 7 /ml, and then 100 ⁇ l/ml of Biotin-TCR antibody (purchased from Meisei, Germany) was added, and incubation was carried out for 10 minutes at 4 ° C in the dark.
  • PBS buffer containing 2 mM EDTA and 1% fetal bovine serum
  • the cell density was adjusted to 1 ⁇ 10 7 cells/ml, and Anti-Biotin Microbeads was added at 50 ⁇ l/ml, and kept at 4 ° C for 15 minutes in the dark.
  • PBS buffer After washing once in PBS buffer, it was resuspended in 500 ⁇ l of buffer.
  • LD column (purchased from Meitian) was placed in a magnetic pole and rinsed with 2 ml of PBS buffer for 1 time. Then, 500 ⁇ l of the cell suspension was added, and the target cells were collected from the bottom of the LD column, and the cells were suspended after the cell suspension was repeated. Add 2 ml of PBS buffer to the LD column. The received cell suspension was centrifuged: 300 g for 5 minutes. Resuspend in pre-warmed medium.
  • Test Example 1 Choosing the best crRNA to CRISPR-Cas9 knockout TRAC
  • the test was compared to the crRNA sequence designed for TRAC shown in Example 5.
  • Cas9 protein was electroporated into activated primary T cells. After 48 hours, the expression of extracellular TCR protein was detected by flow cytometry. The results showed that crRNA can knock out TRAC gene to varying degrees. Among them, crRNA-11 has the highest knockout efficiency.
  • plasmid Three delivery systems: plasmid, mRNA and RNP (protein RNA complex), crRNA for TRAC, plasmid extraction in large quantities.
  • Example 4 for in vitro transcription of Cas9 mRNA.
  • PCR with T7 primer to obtain DNA template containing T7 promoter.
  • the Cas9 mRNA was then transcribed in vitro using Ambion's T7 in vitro transcription kit.
  • the sgRNA and Cas9 protein complexes were obtained in the same manner as in Example 5.
  • Jurkat cells were centrifuged to remove 5 ⁇ 10 6 cells, respectively, and electrotransformed on Invitrogen's electrotransfer system Neon MPK5000 using three different delivery materials.
  • Test Example 3 Random N-oligo or fish sperm DNA increases the efficiency of CRISPR-Cas9 knockout TRAC
  • RNP When a gene knockout is performed using an RNP delivery system, RNP is simultaneously electrotransformed by mixing with a random sequence of N-oligo (oligodeoxyribonucleic acid) or fish sperm DNA.
  • N-oligo sequence An exemplary N-oligo sequence:
  • Example 5 On the basis of Example 5 (3), 100-200 nM of N-oligo DNA was further added to the RNP complex, and the N-oligo DNA was Page grade.
  • the effect of N-oligo on the efficiency of CRISPR-Cas9 knockout TRAC is shown in Figure 2A-2B. The results show that N-oligo can effectively enhance the CRISPR-Cas9 knockout TRAC gene for both T cells and CAR-T cells. s efficiency.
  • Example 5 (3) 100-200 nM fish sperm DNA fragment was further added to the RNP complex, and the effect of the fish sperm DNA fragment on the knockout TRAC efficiency was as shown in Fig. 3, and the result showed that the fish sperm DNA fragment was Increasing the efficiency of TRAC gene knockout is more efficient than N-oligo.
  • Test Example 4 T cell knockout B2M, PD1 efficiency test
  • a number of crRNAs were also designed, and the knockdown of the B2M gene was performed by comparing the crRNA with the highest knockout efficiency and the lowest off-target rate.
  • the B2M and/or PD1 genes of T cells were knocked out using the RNP delivery system and N-oligo based on the same method as in Example 5 (3).
  • the B2M gene expression was closely related to the display of HLA-ABC on the cell membrane, and the knockdown efficiency of the B2M gene was detected using the APC-HLA-ABC antibody (eBioscience).
  • the results show that the knockdown efficiency of the B2M gene is greater than 80%.
  • the RNP and N-oligo mixture were electrotransformed for 48 hours, and 1 ⁇ 10 6 cells were taken separately. After washing twice with PBS buffer, the supernatant was completely aspirated, and the reference kit was used.
  • the Genomic Cleavage Detection Kit (Thermo Fisher) performed the T7E1 experiment. The results (shown in Figure 5) showed that the three crRNAs of the selected PD1 could effectively knock out the PD1 gene, and the knockout efficiency was more than 80%.
  • Test Example 5 Analysis of gene mutations caused by CRISPR-Cas9
  • TRAC TRAC
  • the obtained PCR product DNA fragment was ligated with a T-terminal vector (pEASY-Blunt Simple Cloning Kit, Beijing Quanjin Biotechnology Co., Ltd.). After ligation, TOP10 competent cells were transformed and Amp-resistant solid plates were coated. The next day, clones will be sequenced and at least 30 clones per plate will be tested.
  • Test Example 7 Analysis of the effect of TCR knockdown on cell signaling pathway and killing activity
  • a CD3 antibody solution (5 ⁇ g/ml) was placed in a 96-well plate, and a volume of 100 ⁇ l was added to each well, and coated at 37 ° C for two hours, and after taking out, it was washed twice with PBS. TCR-negative T cells and normal T cells were separately added, and the cell density was 1 ⁇ 10 6 /ml. After incubation at 37 ° C for 24 hours, the flow-through antibodies CD25 and CD69 were taken out, and the results were as shown in Fig. 8A and Fig. 8B. This indicates that T cells cannot be induced to express CD25 and CD69 by CD3 antibodies after TRAC gene knockout.

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109722437A (zh) * 2018-12-29 2019-05-07 广州百暨基因科技有限公司 一种通用型car-t细胞及其制备方法和用途
CN112239769A (zh) * 2019-07-19 2021-01-19 华东师范大学 一种引导PD1基因切割实现外源序列高效整合的sgRNA
WO2021110099A1 (zh) * 2019-12-03 2021-06-10 甘李药业股份有限公司 用于改变基因序列的组合物及方法
WO2021136415A1 (zh) * 2019-12-30 2021-07-08 博雅辑因（北京）生物科技有限公司 一种纯化ucart细胞的方法与应用
US11345932B2 (en) 2018-05-16 2022-05-31 Synthego Corporation Methods and systems for guide RNA design and use

Families Citing this family (3)

* Cited by examiner, † Cited by third party
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EP4039808A1 (en) * 2021-02-08 2022-08-10 Ospedale San Raffaele S.r.l. Guide rnas and uses thereof
CN116987699A (zh) * 2023-09-05 2023-11-03 深圳市艾迪贝克生物医药有限公司 用于制备通用型car-t细胞的基因片段、其工具系统及应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013098244A1 (en) 2011-12-30 2013-07-04 Wageningen Universiteit Modified cascade ribonucleoproteins and uses thereof
WO2013142578A1 (en) 2012-03-20 2013-09-26 Vilnius University RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
CN104395463A (zh) 2012-04-30 2015-03-04 达特茅斯大学理事会 T细胞受体缺陷型t细胞组合物
CN105518146A (zh) 2013-04-04 2016-04-20 哈佛学院校长同事会 利用CRISPR/Cas系统的基因组编辑的治疗性用途
CN106191062A (zh) 2016-07-18 2016-12-07 广东华南联合疫苗开发院有限公司 一种tcr‑/pd‑1‑双阴性t细胞及其构建方法

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206680B (zh) * 2011-04-14 2015-12-16 上海交通大学 基于短链核酸片段的基因电转染缓冲液及其制备和应用方法
RU2685914C1 (ru) * 2013-12-11 2019-04-23 Регенерон Фармасьютикалс, Инк. Способы и композиции для направленной модификации генома
CN103820454B (zh) * 2014-03-04 2016-03-30 上海金卫生物技术有限公司 CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA
KR20230152175A (ko) * 2014-04-18 2023-11-02 에디타스 메디신, 인코포레이티드 암 면역요법을 위한 crispr-cas-관련 방법, 조성물 및 구성성분
CN104328138A (zh) * 2014-09-30 2015-02-04 上海缔达生物科技有限公司 基因组靶标目的基因的定向敲除的方法及试剂盒
EP3212770B1 (en) * 2014-10-29 2022-06-29 Massachusetts Eye & Ear Infirmary Methods for efficient delivery of therapeutic molecules in vitro and in vivo
EP3215166B1 (en) * 2014-10-31 2024-04-24 The Trustees of the University of Pennsylvania Altering gene expression in car-t cells and uses thereof
CN107847524A (zh) * 2015-03-27 2018-03-27 哈佛学院校长同事会 经过修饰的t细胞及其制备和使用方法
WO2016197358A1 (zh) * 2015-06-11 2016-12-15 深圳市第二人民医院 CRISPR-Cas9特异性敲除猪FGL2基因的方法及用于特异性靶向FGL2基因的sgRNA
US20180362975A1 (en) * 2015-12-04 2018-12-20 Novartis Ag Compositions and methods for immunooncology
CN105647871A (zh) * 2016-01-27 2016-06-08 苏州佰通生物科技有限公司 一种可异体移植的嵌合抗原受体t细胞及制备方法
EP3757133A4 (en) * 2018-02-11 2021-12-01 Jiangsu Hengrui Medicine Co., Ltd. ISOLATED CHIMERIC ANTIGEN RECEPTOR, THIS MODIFIED T-CELL CONTAINED AND USES

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013098244A1 (en) 2011-12-30 2013-07-04 Wageningen Universiteit Modified cascade ribonucleoproteins and uses thereof
WO2013142578A1 (en) 2012-03-20 2013-09-26 Vilnius University RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
CN104395463A (zh) 2012-04-30 2015-03-04 达特茅斯大学理事会 T细胞受体缺陷型t细胞组合物
CN105518146A (zh) 2013-04-04 2016-04-20 哈佛学院校长同事会 利用CRISPR/Cas系统的基因组编辑的治疗性用途
CN106191062A (zh) 2016-07-18 2016-12-07 广东华南联合疫苗开发院有限公司 一种tcr‑/pd‑1‑双阴性t细胞及其构建方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAFT ET AL., PLOS COMPUT BIOL., vol. 1, no. 6, 2005, pages e60
LIU, XIAOJUAN ET AL.: "CRISPR-Cas9-Mediated Multiplex Gene Editing in CAR-T Cells", CELL RESEARCH, vol. 27, no. 1, 2 December 2016 (2016-12-02), XP055555205, ISSN: 1001-0602 *
PARK, ROSENBERG ET AL., TRENDS BIOTECHNOL., vol. 29, no. 11, 2011, pages 550 - 557
See also references of EP3650545A4

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11345932B2 (en) 2018-05-16 2022-05-31 Synthego Corporation Methods and systems for guide RNA design and use
US11697827B2 (en) 2018-05-16 2023-07-11 Synthego Corporation Systems and methods for gene modification
US11802296B2 (en) 2018-05-16 2023-10-31 Synthego Corporation Methods and systems for guide RNA design and use
CN109722437A (zh) * 2018-12-29 2019-05-07 广州百暨基因科技有限公司 一种通用型car-t细胞及其制备方法和用途
CN109722437B (zh) * 2018-12-29 2020-01-07 广州百暨基因科技有限公司 一种通用型car-t细胞及其制备方法和用途
CN112239769A (zh) * 2019-07-19 2021-01-19 华东师范大学 一种引导PD1基因切割实现外源序列高效整合的sgRNA
CN112239769B (zh) * 2019-07-19 2023-11-07 上海邦耀生物科技有限公司 一种引导PD1基因切割实现外源序列高效整合的sgRNA
WO2021110099A1 (zh) * 2019-12-03 2021-06-10 甘李药业股份有限公司 用于改变基因序列的组合物及方法
WO2021136415A1 (zh) * 2019-12-30 2021-07-08 博雅辑因（北京）生物科技有限公司 一种纯化ucart细胞的方法与应用

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